1. Field of the Invention
The present invention relates to a method for identifying substances causing differentiation in eukaryotic cells, to DNA constructs, plasmids, viruses and cell lines used in said method, and to a method of preparing a pharmaceutical composition.
2. Description of the Related Art
Differentiation of cells from stem cells is a general biological phenomenon during embryonic development, but also plays a very large part in regeneration processes in the adult organism (e.g., skin regeneration, formation of blood, regeneration of intestinal epithelia, liver regeneration after poisoning or alcohol abuse, etc.). As in all important biological processes, disruptions may cause chronic diseases or may be lethal.
During tumorigenesis, there is very frequently, if not always, a “back”-differentiation of cells, i.e. cells revert to an undifferentiated, embryonic state. Despite great improvements in the methods for early diagnosis and therapy of tumor diseases, mortality is still very high and affected patients suffer immensely. Therefore, finding and developing novel and more effective cancer medicaments (cytostatics) is of extraordinary importance for health policy and have, from the perspective of the pharmaceutical industry, a very large growth potential on the drugs market.
Since many genetic modifications occur on the path from a normal healthy cell to a tumor cell, there are in principle also many possibilities of interfering with the metabolism of tumor cells and thus preventing growth and dissemination. Thus, the largest class of cytostatics leads to damage of cellular DNA. After cell division, this causes lethal mutations, or the cell dies as a direct result of the triggering of the so-called programmed cell death (apoptosis). This affects all rapidly growing cells, i.e. not only tumor cells but also healthy growing cells.
Another class of cytostatics is that of the antimetabolites which halt the metabolism of rapidly growing cells. A third class of cytostatics damages the so-called spindle fiber apparatus of dividing cells, thereby inhibiting cell division or killing the cells directly. A fourth and relatively new class is that of anti-angiogenetic substances which interfere with the ability of tumor cells to induce new blood vessels for their own supply.
A relatively new class of cytostatics, which is relevant in context with the present invention, interferes with the expression pattern of genes which have been partly switched off during tumorigenesis. These are frequently so-called tumor suppressor genes and differentiation genes. The renewed induction and expression of said genes usually leads to a loss of growth advantages of a cancer cell and may also make it easier for the immune system to attack said cell.
The fact that such differentiation-causing substances interfere with the state of methylation of so-called “CpG is-lands” has been known for a long time. CpG islands are found particularly frequently in the 5′-regulatory sequences of genes and play an important part in genomic imprinting, i.e. in regulating paternal versus maternal gene expression. It is possible to switch off genes by methylating the CpG islands present in promoters. If the DNA methylase mainly responsible for methylation is inactive, lethal disruptions in development occur, as can be shown in a knockout model in mice.
It is therefore assumed nowadays that there is a genetically controlled equilibrium of methylation and demethylation in embryogenesis. A disruption of this equilibrium is often present in tumor cells. Thus, it has been found that it is possible to switch off promoters of tumor suppressor genes by methylation of the CpG islands, which is why, for example, the hypomethylation-inducing substance 5′-azacytidine (Aza) acts as a potent cytostatic agent in many types of tumor.
Another mechanism important for differentiation is the influence of histone acetylation. Histones are DNA-binding proteins which can regulate chromatin structure and also influence gene expression. This takes place preferably via biochemical modifications of said histones, for example by acetylation or phosphorylation. Thus, the substance trichostatin A (TSA) is known as a specific inhibitor of histone deacetylase. Recently, it was shown that deacylation of histone H4 leads to chromatin condensation and thereby can suppress gene expression. Accordingly, inhibition of histone deacetylase by TSA leads to chromatin decondensation and can thereby remove suppression of gene expression.
It must be assumed that there are still many other, as yet unknown mechanisms which can be used by tumor cells to switch off expression of differentiation genes and tumor suppressor genes dangerous to them.
While for some classes of cytostatics there are already very good assay systems which also can be used to identify novel compounds, there is, however, a lack of methods for identifying, as mentioned above, such substances capable of causing differentiation. However, this novel class of cytostatics is particularly interesting, because it has great potential for controlling tumors and is associated with substantially fewer side effects than the traditional cytostatics.
The following widespread in vitro test systems are available for identifying substances which cause DNA damage and could therefore potentially be used as cytostatics: the Ames test, or else Salmonella typhimurium test (STY), is based on the mutagenicity of substances in bacteria, while the SOS-Chromotest is based on inducing the bacterial SOS system by genotoxic agents. Both tests have comparable sensitivities, but have the fundamental disadvantage that genotoxic action of substances can vary in bacteria and higher organisms.
For this reason, the Micronucleus test, the single cell gel test (SCG test), also known as comet assay, and the test for sister chromosome exchange (SCE test), which are based on eukaryotic cell systems, have been developed. In the literature, a cell line, A4/4, which contains a lacZ gene under the control of the heavy metal-inducible metallothionein promoter has been described. The authors report that the promoter is switched off during cultivation but can be induced again by the demethylating substance 5′-azacytidine (Biard et al. 1992 Cancer Res 52:5213-5218).
The previously known assay systems for identifying DNA-damaging agents, however, are not suited to identify substances causing differentiation, since the mechanism is completely different.
The reporter cell line described by Biard et al. has the decisive disadvantage of being an inducible system. The demethylating action of substances can be visualized only if the inducer for the promoter is used at the same time. The authors have chosen the metallothionein promoter which is induced by heavy metals such as cadmium and zinc. There are hundreds of indications in the literature for heavy metals themselves inducing gene expression. As a result, the gene expression-causing action of the heavy metals required for the system superimposes the demethylating action of some substances, i.e. unspecific, false negative or false positive results are very easily possible. The cell line was prepared for the purpose of being able to find demethylating substances. These substances may cause differentiation but are, as explained above, not the only substances capable thereof. Since there are, after 1992, no further publications regarding this cell line, it is neither known whether this cell line is stable nor whether it is suitable for detecting other differentiation processes as well.